Calcium signals are vital in controlling cellular events. In smooth muscle cells (SMC), short term Ca2+ signals control contractile responses whereas longer term Ca2+ signals regulate cell growth and proliferation through Ca2+-mediated transcriptional control. The proposed studies focus on the two centrally important Ca2+ signal transducers, STIM1 and STIM2. STIM proteins are finely-tuned endoplasmic reticulum (ER) Ca2+ sensors. STIM proteins are triggered to self-associate and translocate into specialized ER-PM junctions within whom the STIM proteins gate the highly Ca2+ selective store-operated Orai channels. STIM proteins also target voltage-operated L-type Ca2+ channels (LTCC) and exert reciprocal control over these two channel targets, activating Orai but deactivating LTCC, likely important to SMC growth phenotype change in which LTCC is lost and STIM-Orai signaling predominates. While STIM1 has been intensively examined, a major challenge is to understand the role of the poorly-studied STIM2 protein: how differences in its structure, function and expression lead to powerful signaling and phenotypic changes. While whole animal knockout of either STIM1 or STIM2 are lethal, we generated conditional SMC-targeted deletions of STIM1, STIM2, and STIM+STIM2 in mice. The SMC-specific STIM1-KO results in poorly-developed animals dying early, with major defects in the ability of SMC to undergo growth transition, in vivo and in vitro. The SMC conditional STIM1/STIM2 double knockout is perinatally lethal indicating important yet distinct roles of STIM1 and STIM2 in SMC function and growth transition. Using these systems, we have two independent but inter-dependent specific aims 1. To understand the mechanisms and distinctions between STIM1 and STIM2 activation, and to define their molecular coupling to target Ca2+ channels. Using mutational modifications, high resolution Ca2+ and FRET imaging, and electrophysiology, we aim to define the molecular basis of sensing and target channel coupling for STIM1, defining mechanistically important distinctions in the poorly studied STIM2 protein. Building on new structural insights and major functional distinctions between STIM1 and STIM2, our studies focus on defining the interactions through which the two STIM proteins interact with and gate Orai and LTCC target channels. 2. To understand the distinct roles of STIM1 and STIM2 in mediating mitogen-induced growth transition of SMC from the quiescent to proliferative phenotype. Using our SMC-targeted STIM1 and STIM2 null-back- ground mice together with molecular probes to specifically modify STIM1- and STIM2-mediated target channel coupling, we aim to define the differential effectiveness of STIM1 and STIM2 in mediating SMC growth transition. Studies will determine how changes in STIM1 and STIM2 expression and distinctions in their activation and channel coupling, lead to distinct profiles of Ca2+ signals, NFAT activation, and altered growth transition. Our studies apply: (a) new knowledge on the structure and channel-coupling of STIM proteins, (b) innovative STIM1 and STIM2 gene-deletion animal models, (c) innovative probes to assess STIM1 and STIM2 target channel coupling - to provide new understanding of the crucial role of STIM1 in controlling SMC growth transition. Our goals also address a fundamental paucity of information on understanding the mechanism, action and role of STIM2. Information from these studies is crucial to understanding how vascular SMC injury responses occur and how they may be controlled. Our model predicts that distinct patterns of STIM1 and STIM2 expression can be a determining factor in whether SMC undergo phenotype change. Hence the work has fundamental importance in understanding and preventing the SMC growth changes that underlie major vascular diseases including atherosclerosis, hypertension, and arterial restenosis, and SMC phenotype change that underlies lung responses in asthma and angiogenesis that supports growth of tumors.